Effect of nitrogen source on the growth and lipid production of microalgae

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1 Effect of nitrogen source on the growth and lipid production of microalgae H. Varsha rani 1*, K. T.Vijaya Kumar and V. Eswarappa 1 Department of Agricultural Microbiology, University of Agricultural Sciences, Dharwad, Karnataka, India * var_52@rediffmail.com ABSTRACT The growth and lipid production from microalgae was studied under different concentrations of nitrogen sources in BG11 media. The microalgae used in the study are braunii, Scenedesmus dimorphus (SD7), sp (B6). and Neochloris oleoabundans. The different concentration of sodium nitrate varied at 8.5 mm, 17 mm, 34 mm L -1 in BG 11 media. In all the microalgae the highest biomass and lipid production was obtained at the concentration of 8.5 mm of sodium nitrate. The biomass production was maximum in case of braunii (5.00 mg ml -1 ), followed by Scenedesmus dimorphus (4.267 mg ml -1 ) sp. (4.207 mg ml -1 ) and Neochloris oleoabundans (3.567 mg ml -1 ). The strain braunii, at Sodium nitrate of 8.5 mm L -1 yielded highest chlorophyll content of µg ml -1 and decreased to µg ml -1 in Sodium nitrate concentration of 34 mm L -1. The same effect is found in other three algal strains. Similarly, the maximum lipid production in braunii, Neochloris oleoabundans, Scenedesmus dimorphus SD7 and sp. B6 was 2.700, 2.100, and mg ml -1 respectively at 8.5 mm L -1. Then the lipid production was decreased to 2.333, 2.100, and mg ml -1 respectively at 34 mm L -1 concentration of sodium nitrate. There was increase in the biomass and lipid production in all when grown in medium containing sodium nitrate at 8.5 mm L -1 compared to standard BG11 media, where the nitrogen level concentration was 17 mm L -1. KEY WORDS: braunii, microalgae, Neochloris oleoabundans, Scenedesmus dimorphus, sodium nitrate INTRODUCTION high value added foods, pharmaceutical products, or as food for aquaculture etc. The The microalgae are unicellular, lipids from microalgae could be used in photosynthetic organisms similar to plants. different processes for energy exploitation, They also have higher photosynthetic including the simple combustion in boiler or efficiency than plants for the production of in a diesel engine. However, the best biomass. Microalgae are used in different possible use of this oil is certainly its fields such as biofuel production, transformation to a biofuel, especially purification of waste water extractions, biodiesel. Biodiesel has become an important environmental benefits and the 426

2 fact that it is made from renewable resources. A major limitation for microalgal oil production is their relatively high costs, which is expected to be overcome by the technology developments. Process optimization is a very important aspect of such technology development. The biomass productivity, lipid cell content, and overall lipid productivity are some of the key parameters affecting the economic feasibility of algal oil for biodiesel production. Therefore, an ideal process should be able to produce lipid at the highest productivity with the highest lipid cell content. Usually the highest productivity of lipid is obtained by cultivation of the microalgal cells under stress, typically nutrient limitation like nitrogen, iron, calcium, etc,, which is often associated with high biomass and lipid productivity. In the present studies the nitrogen limitation is taken as the main limiting factor for the production of biomass and lipid. MATERIALS AND METHODS The experiment was conducted at AICRP on RES (Bioconversion Technology) Lab, MARS, UAS, Dharwad, Karnataka, India, in the academic year Maintenance of algal culture The strains are separately grown in 100 ml of BG11 medium in 250 ml flask in triplicates for three weeks. These were incubated for the growth in the growth chamber having 25,000 lux light intensity, light and dark cycles (12:12) for three weeks. The homogenization of the cultures is done by using glass beads of 200 mm and the flasks were kept under shaking condition for 20 minutes. The known volume of the cultures are used for the estimation of the biomass, chlorophyll and lipid production Effect of different concentration of sodium nitrate The concentration of sodium nitrate was varied at 8.5 mm, 17 mm, 34 mm in BG 11 media. The microalgal used for the study are braunii, Scenedesmus dimorphus, sp. and Neochloris oleoabundans. The strains were inoculated to these varied medium separately. The growth of microalgae was evaluated in terms of biomass and chlorophyll content and lipid production. Biomass estimation (Richmond and Gobbelaar, 1986). The culture of 100 ml was filtered in a dried and pre-weighed Whatman Filter Paper No.1. This was dried in oven at 60 C until constant weight was obtained. The biomass yield was calculated. Final weight (g) Initial weight (g) Biomass yield (mg ml -1 ) = Sample taken (ml) 427

3 Chlorophyll estimation (Lichtenthaler and Buschmann, 2001) A know volume of homogenized culture was centrifuged at 8000 rpm for 10 minutes and pellet was treated with 10 ml of 95 per cent methanol, shaken well, incubated at 60 C in water bath for 30 minutes. The absorbance of the supernatant was measured in the wave length of and nm in spectrophotometer using 95 per cent of methanol as a blank. Chlorophyll (a) and chlorophyll (b) were estimated as given below. Chlorophyll a (µg ml -1 ) = x A x A Chlorophyll b (µg ml -1 ) = x A x A Total Chlorophyll (a+b) content was calculated. Lipid estimation (Lee et al., 1998). The lipid extraction is done by using chloroform/methanol (2:1) and estimated gravimetrically. Final weight (g) Initial weight (g) Lipid content (mg ml -1 ) = Statistical analysis Sample taken (ml) The data were subjected to Factorial Design analysis and interpretation of the ISSN data was carried out in accordance with Panse and Sukhatme (1985). The level of significance used in the F and t test was P=0.01. The critical difference values were calculated. RESULTS AND DISCUSSION The microalgae braunii, Scenedemus dimorphus (SD7) sp. (B6) and Neochloris oleoabundans grown in 8.5 mm L -1, 17 mm L -1 and 34 mm L -1 concentrations of Sodium nitrate separately. The growth of microalgae was evaluated in terms of biomass and chlorophyll content and lipid production. Effect of different levels of Sodium nitrate on the biomass, chlorophyll content and lipid production of microalgae Among the three levels 8.5 mm L -1, 17 mm L -1 and 34 mm L -1 of sodium nitrate concentrations, 8.5 mm L -1 concentration showed the maximum biomass, chlorophyll and lipid production in all the microalgae. There was significant difference in biomass, chlorophyll and lipid production between the various levels of sodium nitrate. The biomass production was maximum in case of braunii (5.00 mg ml -1 ) at 8.5 mm L -1 of sodium nitrate level which was followed by Scenedesmus dimorphus (4.267 mg ml -1 ) and sp. (4.207 mg ml -1 ) (Table 1). The strain braunii, at Sodium nitrate of 8.5 mm L -1 yielded highest chlorophyll content of µg ml -1 and the chlorophyll content was µg ml -1 in Sodium nitrate concentration of 34 mm L -1. The same effect is found in other three algal strains as shown in Table 2. Similarly, the maximum lipid 428

4 production in braunii, Neochloris oleoabundans, Scenedesmus dimorphus SD7 and sp. B6 was 2.700, 2.100, and mg ml -1 respectively at 8.5 mm L -1. Then the lipid production was decreased to 2.333, 2.100, and mg ml -1 respectively at 34 mm L -1 concentration of sodium nitrate (Table 3). The biomass, chlorophyll and lipid production was decreased in all the microalgal strains as the sodium nitrate concentration was increased in the medium. This effect was due to the non-adaptability of the microalgal strains for high level of Sodium nitrate. A similar study conducted by Li et al. (2008), the highest biomass obtained was 3.15 g L -1 at 10 mm concentration of sodium nitrate. Then the highest lipid produced was 0.4 g g -1 at lower concentration of 3mM L -1 and at higher concentration of 10mM L -1, lipid produced from microalgae was 0.21 g g -1. In another study, Hung and Teng (2009) used urea at varied concentration as nitrogen source. The biomass and lipid production of Chlorella sp. was g L -1 and g g -1 respectively at g L -1 of urea concentration. Then the biomass and lipid production was g L -1 and g g -1 at g L -1 of urea concentration. Illman et al. (2000) found the reduction in nitrogen in the medium increase the lipid content in which Chlorella emersonii, Chlorella nutissima and C. vulgaris gained an increased in lipid content of 63 per cent, 56 per cent and 40 per cent biomass by dry weight respectively. The intracellular lipid content of Nannochloris sp. UTEX LB 1999 grown in a nitrogen limited medium of 0.9 mm KNO 3 concentration was 1.3 times higher than that of which is grown 2.0mM KNO 3 in the medium (Takagi et al., 2000). Sodium nitrate is the limiting factor in protein biosynthesis which inturn increases the lipid /protein ratio. This was reported by Converti et al. (2009). Tornabene et al. (1983) also achieved the highest lipid content of 54 per cent of its biomass with a lower sodium nitrate concentration of 0.5 mm L -1 in Neochloris oleoabundans microalgae. CONCLUSION The sodium nitrate concentration varied at 8.5 mm L -1, 17 mm L -1 and 34 mm L -1 in media. The highest biomass, chlorophyll and lipid production of all the isolates was maximum in 8.5 mm L -1 of the sodium nitrate concentration in the media. braunii and Scenedesmus dimorphus yielded maximum biomass and lipid production at 8.5 mm L -1 sodium nitrate concentration. At the higher concentration of more than 15 mm L -1 caused negative effect on the biomass and lipid production of the microalgal strains. This study concludes the limitation in the nutrient in the media increases the lipid production in the microalgae. The microalge has great potential to provide large quantities of lipids and hydrocarbons that can be converted into biodiesel. The need for renewable biofuels will continue to grow as fossil fuel reserves decline. 429

5 Table 1: Effect of different levels of Sodium nitrate (NaNO 3 ) on the biomass production (mg ml -1 ) by the isolates and standard cultures Sl. No. Concentration of Sodium nitrate (NaNO 3 ) braunii (Ref1) Neochloris oleoabundans (Ref2) Strains Scenedesmus dimorphus (SD7) sp. (B6) Mean mm mm mm Mean Variables S.Em.± 1% Organisms NaNO 3 levels Interactions

6 Table 2: Effect of different levels of Sodium nitrate (NaNO 3 ) on the chlorophyll content (µg ml -1 ) by the isolate of microalgae Sl. No. Concentration of Sodium nitrate (NaNO 3 ) braunii (Ref1) Neochloris oleoabundans (Ref2) Strains Scenedesmus dimorphus (SD7) sp. (B6) Mean mm mm mm Mean Variables S.Em± 1% Organisms NaNO 3 levels Interactions

7 Table 3: Effect of different levels of Sodium nitrate (NaNO 3 ) on the lipid production (mg ml -1 ) by the isolates of microalgae Sl. No. Concentration of Sodium nitrate (NaNO 3 ) braunii (Ref1) Neochloris oleoabundans (Ref2) Strains Scenedesmus dimorphus (SD7) sp. (B6) Mean mm mm mm Mean Variables S.Em. ± 1% Organisms NaNO 3 Levels Interactions

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